Chemical Methods for Producing Disulfide Bonds in Peptides and Proteins to Study Folding Regulation

Abstract

Disulfide bonds play a critical role in the folding of secretory and membrane proteins. Oxidative folding reactions of disulfide bond-containing proteins typically require several hours or days, and numerous misbridged disulfide isomers are often observed as intermediates. The rate-determining step in refolding is thought to be the disulfide-exchange reaction from nonnative to native disulfide bonds in folding intermediates, which often precipitate during the refolding process because of their hydrophobic properties. To overcome this, chemical additives or a disulfide catalyst, protein disulfide isomerase (PDI), are generally used in refolding experiments to regulate disulfide-coupled peptide and protein folding. This unit describes such methods in the context of the thermodynamic and kinetic control of peptide and protein folding, including (1) regulation of disulfide-coupled peptides and protein folding assisted by chemical additives, (2) reductive unfolding of disulfide-containing peptides and proteins, and (3) regulation of disulfide-coupled peptide and protein folding using PDI.

Human ER Oxidoreductin-1α (Ero1α) Undergoes Dual Regulation through Complementary Redox Interactions with Protein-Disulfide Isomerase

“…The digested peptide fragments were analyzed by HPLC (GL Science) equipped with a COSMOSIL 5C 18 -AR-II column (Nacalai Tesque) pre-equilibrated with 5% acetonitrile in 0.05% TFA and eluted with a linear acetonitrile gradient ranging from 5% to 65% at a rate of 1%/min, with detection at an absorbance of 220 nm. The molecular masses of separated peptides were determined by MALDI-TOF MS (Bruker Daltonics) (36,37). Synthetic peptides were prepared by the Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid phase method, modified with AMS, and analyzed by HPLC and MALDI-TOF MS as described previously (5,34,37).…”

“…The molecular masses of separated peptides were determined by MALDI-TOF MS (Bruker Daltonics) (36,37). Synthetic peptides were prepared by the Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid phase method, modified with AMS, and analyzed by HPLC and MALDI-TOF MS as described previously (5,34,37).…”

Antifungal Plant Defensins: Mechanisms of Action and Production

“…Since post-translational modifications mainly occur at the termini of peptides, EPL is a plausible approach to obtain functional peptides [91,92]. When a disulphide bond pattern is essential, (re-)folding of the protein is advised and oxidative folding is performed [93,94,95,96]. In a first attempt, oxidative folding is often employed in a direct manner, i.e.…”

A Novel Toxin from Haplopelma lividum Selectively Inhibits the NaV1.8 Channel and Possesses Potent Analgesic Efficacy

“…In this work, we found that the condition of low pH (6.8) and low temperature (4 °C) were the optimum conditions for refolding of µ-TRTX-Hl1a. A variety of chemical additives, e.g., urea, guanidine hydrochloride (Gu/HCl), and arginine, could improve refolding of disulfide-coupled proteins [ 22 ]. The refolding was increased from 8.52% to 10.38% with the addition of 0.5 M L -Arg, but greatly increased the complexity of refolded protein.…”